Enhanced α-Zearalenol Hydrolyzing Activity of a Mycoestrogen-Detoxifying Lactonase by Structure-Based Engineering

Abstract

The enzyme ZHD101 from Clonostachys rosea hydrolyzes and deactivates the mycotoxin zearalenone (ZEN) and its zearalenol (ZOL) derivatives. ZHD101 prefers ZEN to ZOL as its substrate, but ZOL, especially the α-form, shows higher estrogenic toxicity than ZEN. To enhance α-ZOL selectivity, we solved the complex structures of ZHD101 with both ZOLs and modified several lactone-surrounding residues. Among the mutants, V153H maintained activity for ZEN but showed a 3.7-fold increase in specific activity against α-ZOL, with an ∼2.7-fold reduction in substrate affinity but a 5.2-fold higher turnover rate. We then determined two V153H/ZOL complex structures. Here, the α-ZOL lactone ring is hydrogenbonded to the H153 side chain, yielding a larger space for H242 to reconstitute the catalytic triad. In conclusion, structure-based engineering was successfully employed to improve the ZHD101 activity toward the more toxic α-ZOL, with great potential in further industrial applications.

Detoxification of Mycotoxins through Biotransformation

“…In addition, ZEN (33) was converted to hydrolyzed ZEN (155) through hydrolysis, and then to decarboxylated hydrolyzed ZEN (156) through spontaneous decarboxylation by Bacillus pumilus ( Figure S84) [122]. Similar biotransformation of ZEN (33) was also observed by using a lactonase named ZHD101 from Clonostachys rosea [123,124].…”

“…In the rat liver and intestines, T-2 toxin (31) was first hydrolyzed into HT-2 toxin (122), which was further converted to 15-acety-tetraol (123), which was finally transformed to T-2 tetraol (124) with hydrolyzation ( Figure S61) [102].…”

“…T-2 toxin (31) HT-2 toxin (122) Eubacterium BBSH 797 (bacterium) [86] HT-2 toxin (122),15-acetyl-tetraol (123),T-2 tetraol (124) Liver and intestines of rats [102] Neosolaniol ( Bacillus amyloliquefaciens (bacterium) [98] Lactone-opened ochratoxin A (OP-OTA, 120) Rats [99] Ochratoxin C (OTC) = Ochratoxin A ethyl ester (121) Ochratoxin A (OTA, 21)…”

“…Bacillus pumilus (bacterium) [122] Hydrolysis, spontaneous decarboxylation Lactonase [123] Hydrolysis, spontaneous decarboxylation Lactonase [124] Iso-DOM-3-GlcA (95) and iso-DOM-8-GlcA (96) Isomerization and glucuronization RLM [71] Fomannoxin (40) Fomannoxin acid (59) and fomannoxin acid β-glucoside (145) Oxidation and glycosylation Cell cultures of Pinus sylvestris [44] Fumonisin B1 (115) Hydrolyzed fumonisin B1 = Aminopentol 1 (AP1, 116) and 2-keto AP1 (132)…”

The structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolysis product of zearalenone at 1.60 Å resolution

“…ZHD has demonstrated potential for use as a treatment for ZEN contamination that will not result in damage to cereal crops. Structures of apo ZHD and some enzyme-substrate complexes have recently been reported (Peng et al, 2014;Xu et al, 2016). These structures reveal a catalytic triad consisting of Ser102-His242-Glu126 inside a ZEN-binding tunnel that is enclosed by the / -hydrolase fold and a helical cap domain.…”